THE DETERMINATION OF THE SEQUENCES PRESENT IN THE SHADOW BANDS OF A DINUCLEOTIDE REPEAT PCR

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA re peat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in thi s paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat w as ambiguous. However, the sequence 5' and 3' to the CA repeat was cle ar and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that th e shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly r educe the occurrence of shadow bands.